ABSTRACT Rotavirus infection is a prominent global health concern, causing severe gastroenteritis and infecting nearly every child worldwide by the age of five, despite the currently available vaccines. As a gastrointestinal pathogen, rotavirus primarily infects mature enterocytes in the small intestine where they face a variety of different immune responses mounted by the host. One of these host defense mechanisms is the secretion of antimicrobial peptides. Specifically, our lab is interested in one group of these potent antimicrobial peptides called defensins. Defensins have well-characterized broad antimicrobial activity. We and others have shown that a-defensins can function by multiple mechanisms to neutralize viral infection. Despite this potent antiviral activity, certain viruses can escape or even exhibit enhanced infection in the presence of defensins. One of these viruses is rotavirus. This proposal seeks to determine the exact nature of how rotaviruses have adapted to host defensins to evade neutralization during initial infection and, in some instances, usurp this host defense factor and facilitate infection. I hypothesize that rotavirus exploits the presence of enteric a-defensins during initial infection to enhance cell entry. Aim 1 will establish the specificity of rotavirus adaptation to the presence of a-defensins during infection by testing the effects of homologous (species specific) and heterologous (cross-species) a- defensins on rotavirus infection from 3 different species. Aim 2 will determine the effect that defensins have on rotavirus binding to the cells using fluorescently labeled virus. Aim 3 will define the molecular basis for interaction between a-defensins and rotavirus using an experimental evolution approach and deep genome sequencing to identify regions in rotavirus that are important for interaction with defensin. These findings will be further explored using the novel rotavirus reverse-genetics system. The idea of defensin-mediated enhancement of viral infection is an important component to understanding innate immune system interactions with pathogens. Together these three aims could inform the importance of host factors that contribute to the worldwide burden of diarrheal disease.